Project Summary/Abstract To create a roadmap for rational therapeutic design targeting Ca dysregulation, associated with heart failure and arrhythmia, we seek to understand the protein interactions and structural dynamics that regulate active Ca transport in cardiac muscle. Previous work focused on two membrane proteins, the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA), in both skeletal and cardiac muscle, and its principal cardiac peptide subunit regulator: phospholamban (PLB). The project now focuses on the heart (SERCA2a isoform) and extends to additional peptide regulators. Our core technology is site-directed spectroscopy, in both purified proteins and living cells. We develop and apply innovative and complementary methods in site-directed labeling, fluorescence, EPR, and crystallography, with results integrated by computational simulations and function in biochemical and cellular assays. Aims 1&2 identify fundamental mechanisms, while Aim 3 combines these techniques and resultant insights to develop biophysical assays for therapeutic design. Aim 1 focuses on the functional dynamics of SERCA2a and its key structural transitions that mediate catalytic mechanism, focusing on steps that are critical for regulation in the heart. Added emphasis is now placed on detection of transient structural kinetics, using stopped-flow FRET methods pioneered by our lab, to directly relate SERCA structure and function. Aim 2 investigates mechanisms by which SERCA is regulated by three cardiac peptide subunits: PLB, sarcolipin (SLN), and DWORF. Aim 3 employs the insights of Aims 1&2 and our breakthroughs in high-throughput fluorescence detection, to implement novel small-molecule screening assays, with the ultimate goal of therapeutic discovery for heart disease. New compounds identified in Aim 3 feed back to provide mechanistic insight in Aims 1&2. Thus our Aims are synergistic, strengthening each other with new insights and hypotheses, yet not interdependent, since feasibility has been established independently for each Aim and sub-Aim. This project brings together a powerful and complementary combination of techniques and concepts, from biophysics to chemical biology to molecular genetics to cell biology, performed by a highly-integrated collaborative team, now adding a subcontract (Zima) to further enhance cellular and physiological relevance. The project remains grounded in fundamental biophysical mechanisms, and continues to exploit the recognized value of SERCA Ca pumps as therapeutic targets for major unmet needs in public health, targeting not only the heart, but also skeletal muscle (muscular dystrophy, sarcopenia), neurodegeneration (Alzheimer?s, Parkinson?s), and metabolic disease (diabetes, obesity). Thus, the significance of our work extends well beyond the heart. Our biophysical approaches will surely play a crucial role in understanding SERCA regulation, and also in controlling these functions. Our collaborators and consultants include scientists with expertise in therapeutic development, from academia and industry, and this project continues to stimulate separate efforts in truly translational research. 1